Desaturase antigen of Mycobacterium tuberculosis

ABSTRACT

The present invention relates to the isolation of a new gene, des, which encodes a  M. tuberculosis  protein named DES. The des gene appears to be conserved among different Mycobacteria species. The amino acid sequence of the DES protein contains two sets of motifs that are characteristic of the active sites of enzymes from the class II diiron-oxo protein family. Among this family of proteins, DES shares significant homology with soluble stearoyl-ACP desaturases. DES is a highly antigenic protein, which is recognized by human sera from patients infected with  M. tuberculosis  and  M. leprae  but not by sera from tuberculous cattle. Thus, the DES protein provides a useful tool for the serodiagnostic analysis of tuberculosis.

This is a continuation of application Ser. No. 09/422,662, filed Oct. 22, 1999, now U.S. Pat. No. 6,204,038, which is a divisional of application Ser. No. 08/917,299, filed Jul. 25, 1997, now U.S. Pat. No. 6,010,855, which claims the benefit of U.S. provisional application No. 60/022,713, filed Jul. 26, 1996, all of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Tuberculosis and leprosy, caused by the bacilli from the Mycobacterium tuberculosis complex and M. leprae respectively are the two major mycobacterial diseases. Pathogenic mycobacteria have the ability to survive within host phagocytic cells. From the interactions between the host and the bacteria results the pathology of the tuberculosis infection through the damages the host immune response causes on tissues (Andersen & Brennan, 1994). Alternatively, the protection of the host is also dependent on its interactions with mycobacteria.

Identification of the bacterial antigens involved in these interactions with the immune system is essential for the understanding of the pathogenic mechanisms of mycobacteria and the host immunological response in relation to the evolution of the disease. It is also of great importance for the improvement of the strategies for mycobacterial disease control through vaccination and immunodiagnosis.

Through the years, various strategies have been followed for identifying mycobacterial antigens. Biochemical tools for fractionating and analysing bacterial proteins permitted the isolation of antigenic proteins selected on their capacity to elicit B or T cell responses (Romain et al., 1993; Sorensen et al., 1995). The recent development of molecular genetic methods for mycobacteria (Jacobs et al., 1991; Snapper et al., 1990; Hatful, 1993; Young et al., 1985) allowed the construction of DNA expression libraries of both M. tuberculosis and M. leprae in the λgt11 vector and their expression in E. coli. The screening of these recombinant libraries using murine polyclonal or monoclonal antibodies and patient sera led to the identification of numerous antigens (Braibant et al., 1994; Hermans et al., 1995; Thole & van der Zee, 1990). However, most of them turned out to belong to the group of highly conserved heat shock proteins (Thole & van der Zee, 1990; Young et al., 1990).

The observation in animal models that specific protection against tuberculosis was conferred only by administration of live BCG vaccine, suggested that mycobacterial secreted proteins might play a major role in inducing protective immunity. These proteins were shown to induce cell mediated immune responses and protective immunity in guinea pig or mice model of tuberculosis (Pal & Horwitz, 1992; Andersen, 1994; Haslov et al., 1995). Recently, a genetic methodology for the identification of exported proteins base on PhoA gene fusions was adapted to mycobacteria by Lim et al. (1995). It permitted the isolation of M. tuberculosis DNA fragments encoding exported proteins. Among them, the already known 19 kDa lipoprotein (Lee et al., 1992) and the ERP protein similar to the M. leprae 28 kda antigen (Berthet et al., 1995).

SUMMARY OF THE INVENTION

We have characterized a new M. tuberculosis exported protein named DES identified by using the PhoA gene fusion methodology. The des gene, which seems conserved among mycobacterial species, encodes an antigenic protein highly recognized by human sera from both tuberculosis and leprosy patients but not by se a from tuberculous cattle. The amino acid sequence of the DES protein contains two sets of motifs that are characteristical of the active sites of enzymes from the class II diiron-oxo protein family. Among this family, the DES protein presents significant homologies to soluble stearoyl-ACP desaturases.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with description, serve to explain the principles of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention will be further clarified by the following examples, which are intended to be purely exemplary of the invention.

Bacteria, Media and Growth Conditions

The bacterial strains and plasmids used in this study are listed in FIG. 8. E. coli DH5α or BL21 (DE3)pLysS cultures were routinely grown in Luria B medium (Difco) at 37° C. Mycobacterium cultures were grown in Middlebrook 7H9 medium (Difco) supplemented with Tween 0.05%, glycerol (0.2%) and ADC (glucose, 0.2%; BSA fraction V, 0.5%; and NaCl, 0.085%) at 37° C. Antibiotics when required were added at the following concentrations: ampicillin (100 μg/ml), kanamycin (20 μg/ml).

Human and Cattle Sera

Serum specimens from 20 individuals with pulmonary or extra-pulmonary tuberculosis (M. tuberculosis infected) were obtained from the Bligny sanatorium (France). 6 sera from M. bovis infected human tuberculous patients and 24 sera from BCG-vaccinated patients suffering from other pathologies were repectively obtained from Institut Pasteur, (Madagascar), and the Centre de Biologie Médicale spécialisée (CBMS) (Institut Pasteur, Paris). Sera from tuberculous cattle (M. bovis infected) were obtained from CNEVA, (Maison Alfort).

Subcloning Procedures

Restriction enzymes and T4 DNA ligase were purchased from Gibco/BRL, Boehringer Mannheim and New England Biolabs. All enzymes were used in accordance with the manufacturer's recommendations. A 1-kb ladder of DNA molecular mass markers was from Gibco/BRL. DNA fragments used in the cloning procedures were gel purified using the Geneclean II kit (BIO 101 Inc., La Jolla, Calif.). Cosmids and plasmids were isolated by alkaline lysis (Sambrook et al., 1989). Bacterial strains were transformed by electroporation using the Gene Pulser unit (Bio-Rad Laboratories, Richmond, Calif.).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a restriction map of the 4.5 kb EcoRV fragment encoding the M. tuberculosis des gene.

FIGS. 2A & 2B shows the nucleotide and derived amino acid sequences of the M. tuberculosis des gene.

FIGS. 3A & 3B shows a comparative sequence analysis of class II diiron-oxo proteins and the M. tuberculosis DES protein. Shaded residues indicate cluster ligands and probable iron ligands in the M. tuberculosis DES protein. Bold unshaded framed letters are probable residues involved in the network of hydrogen bonds to the cluster. Other bold letters indicate conserved residues that are believed to participate in the O₂-binding site. Gaps introduced into the sequence of DES are indicated by dots. Accession numbers are as follows: V01555, Epstein-Barr virus ribonucleotide reductase; M58499, Methylococcus capsulatus methane monooxygenase hydroxylase; M60276, Pseudomonas sp. strain CF 600 phenol hydroxylase dmpN polypeptide; M59857, Ricinus communis stearoyl-ACP desaturase; and D38753, O. sativa stearoyl-ACP desaturase.

FIG. 4 is a Southern blot analysis of the distribution of the des gene in other mycobacterial species. DNA from various mycobacterial strains were PstI-digested, electrophoresed, transferred onto a nylon membrane by Southern blotting, and hybridized using probe B, which is shown in FIG. 1.

FIG. 5 shows an SDS-PAGE gel of soluble and insoluble extracts from E. coli expressing the DES protein on plasmid pETdes (I-1718).

FIG. 6 shows the results of ELISAs of the sensitivity of the anitibody response to the DES antigen of human tuberculous and non-tuberculous patients.

FIGS. 7A & 7B shows the nucleotide and derived amino acid sequence of the Mycoplasma tuberculosis des gene. The underlined sequences correspond to the −35 and −10 boxes of the promoter and a Shine Delgarno sequence that corresponds to the putative ribosomal attachment site, respectively. The adenosine labelled “+1” corresponds to the transcription initiation site.

FIG. 8 is a table of the bacterial strains and plasmids used in this application.

FIG. 9 is a Western blot showing the recognition of the purified DES protein by antibodies from M. bovis and M. tuberculosis-infected humans and cattle.

Southern Blot Analysis and Colony Hybridization

DNA fragments for radiolabeling were separated on 0.7% agarose gels (Gibco BRL) in a Tris-borate-EDTA buffer system (Sambrook et al., 1989) and isolated from the gel by using Geneclean II (BIO 101). Radiolabeling was carried out with the random primed labeling kit Megaprime (Amersham) with 5 μCi of (α⁻³²P)dCTP, and nonincorporated label was removed by passing through a Nick Column (Pharmacia). Southern blotting was carried out in 0.4 M NaOH with nylon membranes (Hybond-N+, Amersham) according to the Southern technique (Southern, 1975), prehybridization and hybridization was carried out as recommended byt he manufacturer using RHB buffer (Amersham). Washing at 65° C. was as follows: two washes with 2×SSPE (150 mM NaCl, 8.8 mM NaH₂PO₄, 1 mM EDTA pH 7.4)—SDS 0.1% of 15 minutes each, one wash with 1×SSPE-SDS 0.1% for 10 minutes, two washes with 0.7×SSPE—SDS 0.1% of 15 minutes each. Autoradiographs Were prepared by exposure with X-ray film (Kodak X-Omat at −80° C. overnight. Colony hybrization was carried out using nylon membrane discs (Hybond-N+0.45 μm, Amersham). E. coli colonies adsorbed on the membranes were lysed in a (0.5M NaOH, 1.5M NaCl) solution, before being placed for one minute in a micro-wave oven to fix the DNA. Hybridization and washings were described for the Southern blotting analysis.

DNA Sequencing and Analysis

Sequences of double-stranded plasmid DNA were determined by the dideoxy-chain termination method (Sanger et al., 1977) using the Taq Dye Deoxy Terminator Cycle sequencing Kit (Applied Biosystems), on a GeneAmp PCR System 9600 (Perkin Elmer), and run on a DNA Analysis System-Model 373 stretch (Applied Biosystems). The sequence was assembled and processed using DNA strider™ (CEA, France) and the University of Wisconsin Genetics Computer Group (UWGCG) packages. The BLAST algorithm (Altschul et al., 1990) was used to search protein data bases for similarity.

Expression and Purification of the DES Protein in E coli

A 1043 bp NdeI-BamHI fragment of the des gene was amplified by PCR using nucleotides JD8 (SEQ ID NO:1) 5′-CGGCATATGTCAGCCAAGCTGACCGACCTGCAG-3′) and JD9 (SEQ ID NO:2) (5′-CCGGGATCCCGCGCTCGCCGCTCTGCATCGTCG3′), and cloned into the NdeI-BamHI sites of pET14b (Novagen) to generate pET-des. PCR amplifications were carried out in a DNA thermal Cycler (Perkin Elmer), using Taq polymerase (Cetus) according to the manufacturer's recommendations. PCR consisted of one cycle of denaturation (95° C., 6 min) followed by 25 cycles of amplication consisting of denaturation (95° C., 1 min), annealing (57° C., 1 min), and primer extension (79° C., 1 min). In the pET-des vector, the expression of the des gene is under control of the T7 bacteriophage promoter and the DES antigen is expressed as a fusion protein containing six histidine residues. Expression of the des gene was induced by addition of 0.4 mM IPTG in the culture medium. The DES protein was purified by using a nickel-chelate affinity resin according to the recommendatins of the supplier (Qiagen, Chatsworth, Calif.). Linked to the localization of the DES protein in cytoplasmic inclusion bodies, the purification was carried out under denaturing conditions in guanidine hydrochloride buffers. The protein was eluted in buffer A (6 M guanidine hydrochloride. 0.1 M NaH₁PO₄, 0.01 M Tris, pH 8) containing 100 mM EDTA. The purified protein was kept and used in buffer A, as all attempts to solubilize it in other buffers were unsuccessful.

SDS-PAGE and Immunoblotting

Sodium dodecyl sulfate-polyacrlamide gel electrophoresis (SDS-PAGE) was carried out as described by Laemmli (1970). For Western blotting experiments (immunoblotting), approximately 10 μg of DES purified protein were run on a SDS-polyacrylamide gel and transferred onto nitrocellulose membranes (Hybond C extra, Amersham) using a Bio-Rad mini transblot apparatus according to the recommendations of the manufacturer (Bio-Rad Laboratories, Richmond, Calif). Transfer yield was visualized by transient staining with Ponceau Rouge. The membrane were incubated with human patient or cattle sera diluted 1/200 at 37° C. for 1 hour and with a goat anti-human (Promega) or rabbit anti-cattle (Biosys) IgG alkaline phosphatase-conjugated secondary antibody diluted 1/2500′ for 30 minutes at 37° C. The color reaction was performed by addition of 5-bromo-4-chloro-3-indolyllphosphate (0.165 mg/ml) and toluidinum nitroblue tetrazolium (0.33 mg/ml) as substrates.

ELISA

The human or cattle sera were tested for antibodies against DES by enzyme-linked immunosorbent assay (ELISA). The 96-well micro-titer trays (Nune) were coated with 0.1 μg per well) of purified DES protein in guanidine hydrochloride buffer A (6 M guanidine hydrochloride, 0.1 M NaH₂PO₄, 001 M Tris, pH 8) (1 h at 37° C. and 16 h at 4° C.). After three washes, wells were saturated with bovine serum albumin 3% in phosphate buffered saline (PBS) for 30 mn at room temperature. After three washes, sera diluted from 1/50° to 1/3200° in buffer (PBS, 0.1% Tween 20, 1% bovine serum albumin) were added to the wells for 2 h at 37° C. After three washes, the wells were treated with goat anti-human IgG-alkaline phosphatase conjugate (promega) diluted 1/4000° for 1 h at 37° C. Then, 4 mg of p-nitrophenylphospate per ml were added as substrate. After 20 mn of incubation at 37° C., the plates were read photometrically at an optical density of 405 nm in micro-ELISA Autoreader (Dynatech, Mames la Coquette, France).

Statistics

Antibody responses of the different sera tested were compared by using the Student t test. P≧0.05 was considered nonsignificant.

Nucleotide Sequence and Accession Number

The nucleotide sequences of des has been deposited in the Genome Sequence Data Base (GSDB) under the accession number U49839.

Cloning of the Des Gene

The construction of a library of fusion of M. tuberculosis genomic DNA to the phoA gene and its expression in M. smegmatis, described by Lim et al. (1995), led to the isolation of several PhoA⁻ clones. pExp421 is the plasmid harboured by one of the PhoA⁺ clones selected from this library. Detection of enzymatically active alkaline phosphatase indicated that the pExp421 insert contains functional expression and exportation signals. Restriction analysis showed that pExp421 carries a 1.1 kb insert. Partial determination of its sequence identified a 577 bp ORF, named des, fused in frame to the phoA gene and presenting two motifs, of 9 and 14 amino acids, conserved with soluble stearoyl-acyl-carrier protein desaturases (Lim et al., 1995).

To isolate the full-length des gene, the M. tuberculosis H37R^(V) pYUB18 genomic cosmid library (Jacobs et al., 1991), was screened by colony hybridization with the 1.1 kb probe (probe A, see FIG. 1). Two hybridizing cosmids named C₃ and C₄ were selected for further isolation of the gene. C₃ and C₄ were cut with several restriction enzymes and subjected to Southern blot analysis using the 1.1 kb fragment as a probe.

The EcoR^(V) restriction profile revealed a single hybridizing fragment of 4.5 kb which was subcloned into pBluescript KS⁻ (Stratagéne) to give plasmid pBS-des.

Characterization of the Des Gene

The DNA sequence of the full des ORF was determined (FIG. 2). The des gene was shown to cover a 1017 bp region, encoding a 339 amino acid protein with a calculated molecular mass of 37 kDa. The ORF starts with a potential ATG start codon at position 549, and ends with a TAG stop codon at position 1565. There is a potential Shine-Dalgamo motif (GGAGG) at position −8 upstream of the ATG. The G+C content of the ORF (62%) is consistent with the global GC content observed mycobacterial genome. The nucleotide and deduced amino acid sequences of the des gene were compared to sequences in databases. They showed very high homologies to the M. leprae aadX gene located on cosmid B2266, deposited in GenBank as part of the M. leprae genome sequencing project (GenBank accession number n^(o). U15182). Within the coding region, the DNA sequences were 79% identical while the encoded proteins were 80% identical (88% including conserved residues). The des gene also scored significantly against soluble stearoyl-ACP desturases: 44% identity at the nucleotide level, 30% identity (51% including conserved residues) at the amino acid level, to the Oryza saliva stearoyl-ACP desaturase (accesion n^(o). D38753).

Althought the detection of a phoA enzymatical activity in the M. smegmatis clone harbouring the pEXp421 suggests the DES protein is exported, no structural similarities were found between the DES protein N terminal amino acids and signal sequences of bacterial exported proteins (Izard & Kendall, 1994).

Like in M. leprae genome, a second ORF presenting high homologies ot the M. lepra putative NtrB gene (cosmid B2266), is located downstream of the des gene in M. tuberculosis FIG. 2. Interestingly, the two ORF, des and <<NtrB>>, are separated in M. tuberculosis by two direct repeats of 66 nucleotides overlapping on 9 nucleotides (FIG. 2). Although M. leprae and M. tuberculosis seem to share the same genomic organization in this part of the chromosome, these repeats from the M. leprae genome.

The Des Protein Presents the Conserved Amino Acid Motifs of the Class II Diiron-oxo Proteins

Further analysis of the amino-acid sequence of the DES protein revealed the presence of conserved motifs found only in class II diiron-oxo proteins (Fox et al., 1994) (FIG. 3). These proteins are oxo-bridged diiron clusters (Fe—O—Fe) containing proteins. They possess in their secondary structure 4 alpha helices involved in the protein-derived cluster ligands. As revealed by X-ray structure studies, in these proteins, the diiron axis is oreiented parallel to the long axis of the four helix bundle with ligands arising from four noncontiguous helices, B, C, E and F. M. tuberculosis DES protein appears to have the same active site residues as the class II diiron-oxo enzymes. This includes Glu and His residues (E₁₀₇ and H₁₁₀ in helix C, E₁₆₇ in helix E and E₁₉₇ and H₂₀₀ in helix F) that are ligands to the iron atoms, Asp, Glu and Arg residues E₁₀₆ and R₁₀₉ in helix C, D₁₉₆ in helix F) that are involved in a hydrogen-bonding network to the cluster and, De and Thr resideues that may be part of the O₂-binding site (T₁₇₀ in helix E, I₁₉₃ in helix F). Thus, the M. tuberculosis DES protein contains in its primary sequence two conserved D/E(ENXH) motifs separated by 85 amino acids.

The class II diiron-oxo protein family contains up to date ribonucleotide reductases, hydrocarbon hydroxylases (methane monooxygenase, toluene-4-monooxygenase and phenol hydroxylase) and soluble-ACP desaturases. On the overall sequence alignment the DES protein presents higher homology to soluble stearoyl-ACP desaturases than to ribonucleotide reductases or bacterial hydroxylases. The percentage identity at the amino acid level of the DES protein was said to be 30% with the Oryza sativa stearoyl-ACP desaturases, whereas it is only 17% with the Methylococcus capsulatus methane monooxygenase (accession n^(o).M58499), 17.5% with the Pseudomonas sp CF 600 phenol hydroxlase (accession n^(o).M60276) and 17.7% with the Epstein Barr ribonucleotide reductase (accession n^(o).^(V)01555). Homologies to the soluble Δ9 desaturases mostly concern the amino acids located within the active site in helices C, E and F (FIG. 3).

Distribution of the Des Gene in Other Mycobacterial Species

The presence of the des gene in PstI-degested chromosomal DNA from various mycobacterial strains was analyzed by Southern blotting (FIG. 4). The probe used (probe B) is a PCR amplication product corresponding to nucleotides 572 to 1589 (see FIG. 1). The probe hybridized on all mycobacterial genomic DNA tested. Strong signals were detected in M. tuberculosis, M. bovis, M. bovis BCG, M. Africanum and M. avium. Weaker signals were visible in M. microti, M. zenopi, M. fortuitum and M. smegmatis. Thus, the des gene seems to be present in single copy at least in the slow growing M. tuberculosis, M. bovis, M. bovis BCG, M. Africanum, M. avium and M. zenopi as well as in the fast growing M. smegmatis.

Expression of the Des Gene in E. coli

In order to overexpress the DES protein, the des gene was subcloned into the bacteriophage T7 promoter-based expression vector pET14b (Novagen). A PCR amplification product of the des gene (see material and methods) was cloned into the NdeI-BamHI sites of the vector, leading to plasmid pET-des. Upon IPTG induction of E. Coli BL21 DE3 pLysS cells harbouring the plasmid pET-des, a protein of about 40 kDa was overproduced. The size of the overproduced protein is in agreement with the molecular mass calculated from the deduced polypeptide. As shown in FIG. 5, the great majority, of the overproduced DES protein is present in the insoluble matter of E. coli cells. This probably results from the precipitation of the over-concentrated protein in E. coli cytoplasm thus forming inclusion bodies. To be able to dissolve the protein, the purification was carried out using a nickel chelate affinity resin under denaturing conditions in guanidine hydrochloride buffers. Among all the conditions tested (pH, detergents . . . ), the only condition in which the protein could be eluted without precipitating in the column and remain soluble, was in a buffer containing 6 M guanidine hydrochloride.

Immunogenicity of the DES Protein After Infection

20 serum samples from M. tuberculosis infected human patients (4 with extra-pulmonary tuberculosis, 15 with pulmonary tuberculosis and 1 with both forms if the disease), 6 sera from M. bovis infected human patients and 4 sera from M. bovis infected cattle were tested either pooled or taken individually in immunoblot experiments to determine the frequency of recognition of the purified DES protein by antibodies from infected humans or cattle. 20 out of the 20 sera from the M. tuberculosis infected human patients and 6 out of the 6 sera from the M. bovis infected human patients recognized the recombinant antigen as shown by the reaction With the 37 kDa band, (FIG. 9). Furthermore, a pool of sera from human lepromatous leprosy patients also reacted against the DES antigen.

In contrast, the pool of serum specimens from M. bovis infected cattle did not recognize the DES protein. These results indicate that the DES protein is highly immunogenic in tuberculosis human patients. Both pulmonary and extra-pulmonary tuberculosis patients recognize the antigen.

Magnitude of Human Patients Antibody Responses

An enzyme-linked immunosorbent assay (ELISA) was used to compare the sensitivity of the different serum samples from 20 tuberculosis patients (15 infected by M. tuberculosis and 5 infected by M. Bovis) to the DES antigen. This technique was also carried out to compare the sensitivity of the antibody response to DES of the 20 tuberculosis patients to the one of 24 patients (BCG-vaccinated) suffering from other pathologies. As shown on FIG. 6, patients suffering from other pathologies than tuberculosis, react at low level to the DES antigen (average OD₄₀₅=0.17 for a serum dilution 1/100⁴). The average antibody response from the tuberculosis patients infected by M. tuberculosis or M. bovis against the same antigen is much more sensitive (OD₄₀₅=0.32 and OD₄₀₅=0.36 respectively, for a serum dilution 1/100⁴). This difference in the sensitivity of the immunological response is statistically highly significant at every dilution from 1/50^(a) to 1/3200^(a) as shown by a Student I₉₅ test (I₉₅=5.18, 6.57, 6.16, 5.79, 4.43, 2.53 and 1.95, at sera dilutions 1/50^(a), 1/100^(a), 1/400^(a), 1/800^(a), 1/600^(a) and 1/3200^(a), respectively). No differences in the sensitivity of the antibody response was noticed between patents suffering from pulmonary or extra-pulmonary tuberculosis.

The Pho A gene fusion methodology permitted the identification of a new M. tuberculosis exported antigenic protein. This 37 kDa exported protein contains conserved amino acid residues which are characteristical of class II diiron-oxoproteins. Proteins from that family are all enzymes that require iron for activity. They include ribonucleotide reductases, hydrocarbon hydroxylases and stearoly-ACP desaturases. The M. tuberculosis DES protein only presents significant homologies to plant stearoyl-ACP desaturases (44% identity at the nucleotide level, and 30% identity at the amino-acid level) which are exported enzymes as they are translocated across the chloroplastic membranes (Keegstra & Olsten, 1989). This result suggests that the DES protein could be involved in the mycobacterial fatty acid biosynthesis. Furthermore, the localization of the protein outside the cytoplasm would be consistent with its role in the lipid metabolism, since lipids represent 60% of the cell wall constituents and that part of the biosynthesis of the voluminous mycolic acids containing 60 to 90 carbon atoms occurs outside the cytoplasm. Among all the different steps of the lipid metabolism, desaturation reactions are of special interest, first because they very often take place at early steps of lipid biosynthesis and secondly because, through the control they have on the unsaturation rate of membranes, they contribute to the adaptation of mycobacteria to their environment (Wheeler & Ratledge, 1994). An enzyme system involving a stearoyl-Coenzyme A desaturase (analog of the plant stearoyl-ACP-desaturases), catalyzing oxydative desaturation of the CoA derivatives of stearic and palmitic acid to the corresponding Δ9 monounsaturated fatty acids has been biochemically characterized in Mycobacterium phlei (Fulco & Bloch, 1962; Fulco & Bloch, 1964; Kashiwabara et al., 1975; Kashiwabara & Sato, 1973). This system was shown to be firmly bound to a membranous structure (Fulco & Bloch, 1964). Thus, M. tuberculosis steroyl-Coenzyme A desaturase (Δ9 desaturase) is expected to be an exported protein. Sonicated extracts of E. coli expressing the DES protein were assayed for Δ9 desaturating activity according to the method described by Legrand and Bensadoun (1991), using (steroyl-CoA) ¹⁴C as a substrate. However, no Δ9 desaturating activity could be detected. This result is probably linked to the fact that desaturation systems are multi-enzyme complexes involving electron transport chains and numerous cofactors, often difficult to render functional in vitro. E. coli and mycobacteria being very different from a lipid metabolism point of view, the M. tuberculosis recombinant Δ9 desaturase might not dispose in E. coli of all the cofactors and associated enzymes required for activity or might not interact properly with them. Moreover, not all cofactors involved in the Δ9 desaturation process of mycobacteria are known, and they might be missing in the incubation medium.

However, if the DES protein encodes a Δ9 desaturase, an amazing point concerns its primary sequence. Indeed, all animal, fungal and the only two bacterial Δ9 desaturases sequenced to date (Sakamoto et al., 1994) are integral membrane proteins which have been classified into a third class of diiron-oxo proteins on the basis of their primary sequences involving histidine conserved residues (Shanklin et al., 1994). The plant soluble Δ9 desaturases are the only desaturases to possess the type of primary sequence of class II diiron-oxo proteins (Shanklin & Somerville, 1991). No bacteria have yet been found which have a plant type Δ9 desaturase.

As shown by immunoblotting and ELISA experiments, the DES protein is a highly immunogenic antigen which elicits B cell response in 100% of the tuberculosis M. bovis or M. tuberculosis-infected human patients tested, independently of the form of the disease (extrapulmonary or pulmonary). It also elicits an antibody response in lepromatous leprosy patients. Interestingly, although more sera would need to be tested, tuberculous cattle do not seem to recognize the DES antigen. Furthermore, the ELISA experiments showed that it is possible to distinguish tuberculosis patients from patients suffering from other pathologies on the basis of the sensitivity of their antibody response to the DES antigen. The DES antigen is therefore a good candidate to be used for serodiagnosis of tuberculosis in human patients. The reason why the non-tuberculous patients tested recognize at a low level the DES protein could be due to the fact they,are all BCG-vaccinated individuals (BCG expressing the protein), or to a cross-reactivity of their antibody response Keith other bacterial antigens. It would now be interesting to know whether he DES antigen possesses in addition to its B cell epitopes, T cell epitopes which are the only protective ones in the host immunological response against pathogenic mycobacteria. If the DES protein is also a good stimulator of the T cell response in a majority of tuberculosis patients, it could be used either individually or as part of a <<cocktail>> of antigens in the design of a subunit vaccine against tuberculosis.

The references cited herein are listed on the following pages and are expressly incorporated by reference.

Other embodiments of the invention trill be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

1. Altschul, S. F., W. Gish, W. Miller, E. M. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. Journal of Molecular Biology. 215:403-410.

2. Anderson, A. B., and B. Brennan. 1994. Proteins and antigens of Mycobacterium tuberculosis, p. 307-332. In B. R. Bloom (ed.), Tuberculosis: Pathogenesis, Protection, and Control. ASM, Washington, D.C.

3. Andersen, P. 1994. Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins. Infect. Immun. 62:2536-2544.

4. Berthet, F. X., J. Rauzier, E. M. Lim, W. Philipp, B. Giequel, and D. Portnoï. 1995. Characterization of the M. tuberculosis erp gene encoding a potential cell surface protein with repetitive structures, Microbiology. 141:2123-2130.

5. Braibant, M., L. D. Wit, P. Peirs, M. Kalai, J. Ooms, A. Drowart, K. Huygen, and J. Content. 1994. Structure of the Mycobacterium tuberculosis antigen 88, a protein related to the Escherichia coli PstA periplasmic phosphate permease subunit. Infection and Imununity. 62:849-854.

6. Fox, B. G., J. Shanklin, J. Ai, T. M. Loerh, and J. Sanders-Loerb. 1994. Resonance Raman evidence for an Fe—O—Fe center in stearoyl-ACP desaturase. Primary sequence identity with other diiron-oxo proteins. Biochemistry. 33:12776-12786.

7. Fulco, A. J., and. K. Bloch. 1962. Cofactor requirements for fatty acid desaturation in Mycobacterium phlei. Biochim. Biophys. Acta. 63:545-546.

8. Fulco, A. J., and K. Bloch. 1964. Cofactor requirements for the formation of Δ9 unsaturated fatty acids in Mycobacterium phlei. The Journal of Biological Chemistry. 239-993-997.

9. Haslov, K., A. Andersen, S. Nagai, A. Gortschau. T. Sorensen, and P. Andersen. 1995. Guinea pig cellular immune responses to proteins secreted by Mycobacterium tuberculosis. Infection and Immunity. 63:804-810.

10. Hatfull, G. F, 1993. Genetic transformation of mycobacteria. Trends in microbiology. 1:310-314.

11. Hermans. P. W. M., F. Abebe, V. I. O. Kuteyi, A. H. J. Kolk, J. E. R. Thole, and M. Harboe. 1995. Molecular and immunological characterization of the highly conserved antigen 84 from Mycobacterium tuberculosis and Mycobacterium leprae. Infection and Immunity. 63:954-960.

12. Izard J. W., and D. A. Kendall. 1994. Signal peptides: exquisitely designed transport promoters. Molecular Microbiology. 13:765-773.

13. Jacobs, W. R., G. V. Kalpana, J. D. Cirillo, L. Pascopella, S. B. Snapper, R. A. Udani, W. Jones, R. G. Barletta, and B. R. Bloom. 1991. Genetic systems for mycobacteria. Methods enzymol. 204:537-555.

14. Kasbiwabara, Y., H. Nakagawa, G. Matsuki, and R. Sato. 1975. Effect of metal ions in the culture medium on the stearoyl-Coenzyme A desaturase activity of Mycobacterium phlei. J. Biochem. 78:803-810.

15. Kashiwabara, Y., and R. Sato. 1973. Electron transfer mechnism involved in stearoyl-coenzyme A desaturation by particulate fraction of Mycobacteriun phlei. J. Biochem. 74:405-413.

16. Keegstra, K., and L. J. Olsen. 1989. Chloroplastic precursors and their transport across the envelope membranes. Ann. Rev. Plant Physiol. Plant Mol. Biol. 40:471-501.

17. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London). 227:680-685.

18. Lee, B. Y., S. A. Hefta, and P. J. Brennan. 1992. Characterization of the major membrane protein of virulent Mycobacterium tuberculosis. Infection and Immunity. 60:2066-2074.

19. Legrand, P., and A. Bensadoun. 1991. Stearyl-CoA desaturase activity in cultured rat hepatocytes. Biochimica et Biophysica Acia. 1086:89-94.

20. Lim, E. M., J. Rauzier, J, Timm, G. Torrea, A. Murray, B. Gicquel, and D. Portnoï. 1995. Identification of Mycobacterium tuberculosis DNA sequences encoding exported proteins by using phoA gene fusions. Journal of Bacteriology. 177:59-65.

21. Pal, P. G., and M. A. Horwitz: 1992. Immunization with extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substential protective immunity in a guinea pig model of pulmonary tuberculosis. Infection and Immunity. 60:4781-4792.

22. Romain, F., A. Laqueyrerie, P. Militzer, P. Pescher, P. Chavarot, M. Lagranderie, G. Auregan, M. Gheorghiu, and G. Marchal. 1993. Identification of a Mycobacterium bovis BCG 45/47-kilodalton antigen complex, an immunodominant target for antibody response after immunization with living bacteria. Infection and immunity. 61:742-750.

23. Sakamoto, T., H. Wada, I. Nishida, M. Ohmori, and N. Murata. 1994. Δ9 acyl lipid desaturases of cyanobacteria. J. Biol. Chem. 269:25576-25580.

24. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning—A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

25. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing, with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA. 74:5463-5467.

26. Shanklin, J., and C. Somerville. 1991. Stearoyl-acyl-carrier-protein desaturase from higher plants is struturally unrelatad to the animal and fungal homologs. Proceeding of the National Academy of Science of the United States of America. 88:2510-2514.

27. Shanklin, J., E. Whittle, and B. G. Fox. 1994. Eight histidine residues art catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase. Biochemistry. 33:12787-12794.

28. Snapper, S. B., B. R. Bloom, and J. W. R. Jacobs. 1990. Molecular genetic approaches to mycobacterial investigation. p. 199-218. In J. McFadden (ed.), Molecular Biology of the Mycobacteria. Surrey University Press, London.

29. Sorensen, A. L., S. Nagai, G. Houen, P. Andersen, and A. B. Andersen. 1995. Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis. Infection and Immunity. 63:1710-1717.

30. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517.

31. Studier, W., A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods in Enzymology. 185:60-89.

32. Thole, J. E. R., and R. v. d. Zee. 1990. The 65 kD antigen: molecular studies on a ubiquitous antigen., p. 37-66. In J. McFadden (ed.). Molecular Biology of the mycobacteria. Surrey University Press. London.

33. Wheeler, P. R., and C. Ratledge. 1994. Metabolism of Mycobacterium tuberculosis, p. 353-385. In B. R. Bloom (ed.), Tuberculosis: Pathogenesis, Protection, and Control. ASM, Washington, D.C.

34. Young, D., T. Garbe, R. Lathigra, and C. Abou-Zeid. 1990. Protein antigens: structure, function and regulation, p. 1-35. In J. McFadden (ed.). Molecular biology of mycobacteria. Surrey university Press, Laudon.

35. Young, R. A., B. R. Bloom, C. M. Grossinsky, J. Ivany, D. Thomas, and R. W. Davis. 1985. Dissection of the Mycobacterium tuberculosis antigens using recombinant DNA. Proc. Natl. Acad. Sci.USA. 82:2583-2587.

37 33 base pairs nucleic acid single Not Relevant other nucleic acid /desc = “NUCLEIC ACID” 1 CGGCATATGT CAGCCAAGCT GACCGACCTG CAG 33 33 base pairs nucleic acid single Not Relevant other nucleic acid /desc = “NUCLEIC ACID” 2 CCGGGATCCC GCGCTCGCCG CTCTGCATCG TCG 33 1691 base pairs nucleic acid single Not Relevant other nucleic acid /desc = “NUCLEIC ACID” CDS 549..1562 3 GATCATCATC GGCCGGCTGC CGCGCAGGGC GCCGACACCG GCGAGTGCGG GCGCGAGGAT 60 CGGCCCCCAC CAGTTCGGCA GCTGCGTGTC GATGCGCTCC ACAATCCCGG GAAACAGCTC 120 GACCATTACC TCCTCAATAT GAGCCTCGAA AAACTTGCCG CTGTGCGCGG CGTCGTGGTG 180 AGCGCACACA ACAACTGTTA GCTGACCAGC AGGATCGGCG CTCTTACCGG TCTGTTCACC 240 GCATATCTGA ACGGACGGCT GGGAGCCACC CGCAAGCAAT TCATCGACTA CTGCGTCAAC 300 ATGTTGCTCA GCACCGCCGC CACCTACGCA CCGCACCGCG AGCGGGGAGA ATCCGAACAC 360 TCCATCCCAG CCGGGCCGCA CAACTGAGGA CGACTGGGGT TCACCCCACG CGGCCACCGG 420 GGCCCGCCGA TGCCAGCATC CTGCCCGCTG CTGGCAGCTC AACATGCCGC GCGAAGCCCA 480 AACTTGATGC TACCGAGAGA CACAGATATA TTGACTGCAA CCATTAGACA CAGATAACTG 540 GAGGCGCC ATG TCA GCC AAG CTG ACC GAC CTG CAG CTG CTG CAC GAA CTT 590 Met Ser Ala Lys Leu Thr Asp Leu Gln Leu Leu His Glu Leu 1 5 10 GAA CCG GTC GTC GAG AAG TAC CTG AAC CGG CAC CTG AGC ATG CAC AAG 638 Glu Pro Val Val Glu Lys Tyr Leu Asn Arg His Leu Ser Met His Lys 15 20 25 30 CCC TGG AAC CCG CAC GAC TAC ATC CCG TGG TCG GAC GGG AAG AAC TAC 686 Pro Trp Asn Pro His Asp Tyr Ile Pro Trp Ser Asp Gly Lys Asn Tyr 35 40 45 TAC GCG CTC GGC GGG CAG GAT TGG GAC CCC GAC CAG AGC AAG CTT TCT 734 Tyr Ala Leu Gly Gly Gln Asp Trp Asp Pro Asp Gln Ser Lys Leu Ser 50 55 60 GAT GTC GCC CAG GTG GCG ATG GTG CAG AAC CTG GTC ACC GAG GAC AAC 782 Asp Val Ala Gln Val Ala Met Val Gln Asn Leu Val Thr Glu Asp Asn 65 70 75 CTG CCG TCG TAT CAC CGC GAG ATC GCG ATG AAC ATG GGC ATG GAC GGC 830 Leu Pro Ser Tyr His Arg Glu Ile Ala Met Asn Met Gly Met Asp Gly 80 85 90 GCG TGG GGG CAG TGG GTC AAC CGT TGG ACC GCC GAG GAG AAT CGG CAC 878 Ala Trp Gly Gln Trp Val Asn Arg Trp Thr Ala Glu Glu Asn Arg His 95 100 105 110 GGC ATC GCG CTG CGC GAC TAC CTG GTG GTG ACC CGA TCG GTC GAC CCT 926 Gly Ile Ala Leu Arg Asp Tyr Leu Val Val Thr Arg Ser Val Asp Pro 115 120 125 GTC GAG TTG GAG AAA CTT CGC CTC GAG GTA GTC AAC CGG GGC TTC AGC 974 Val Glu Leu Glu Lys Leu Arg Leu Glu Val Val Asn Arg Gly Phe Ser 130 135 140 CCA GGC CAA AAC CAC CAG GGC CAC TAT TTC GCG GAG AGC CTC ACC GAC 1022 Pro Gly Gln Asn His Gln Gly His Tyr Phe Ala Glu Ser Leu Thr Asp 145 150 155 TCC GTC CTC TAT GTC AGT TTC CAG GAA CTG GCA ACC CGG ATT TCG CAC 1070 Ser Val Leu Tyr Val Ser Phe Gln Glu Leu Ala Thr Arg Ile Ser His 160 165 170 CGC AAT ACC GGC AAG GCA TGT AAC GAC CCC GTC GCC GAC CAG CTC ATG 1118 Arg Asn Thr Gly Lys Ala Cys Asn Asp Pro Val Ala Asp Gln Leu Met 175 180 185 190 GCC AAG ATC TCG GCA GAC GAG AAT CTG CAC ATG ATC TTC TAC CGC GAC 1166 Ala Lys Ile Ser Ala Asp Glu Asn Leu His Met Ile Phe Tyr Arg Asp 195 200 205 GTC AGC GAG GCC GCG TTC GAC CTC GTG CCC AAC CAG GCC ATG AAG TCG 1214 Val Ser Glu Ala Ala Phe Asp Leu Val Pro Asn Gln Ala Met Lys Ser 210 215 220 CTG CAC CTG ATT TTG AGC CAC TTC CAG ATG CCC GGC TTC CAA GTA CCC 1262 Leu His Leu Ile Leu Ser His Phe Gln Met Pro Gly Phe Gln Val Pro 225 230 235 GAG TTC CGG CGC AAA GCC GTG GTC ATC GCC GTC GGG GGT GTC TAC GAC 1310 Glu Phe Arg Arg Lys Ala Val Val Ile Ala Val Gly Gly Val Tyr Asp 240 245 250 CCG CGC ATC CAC CTC GAC GAA GTC GTC ATG CCG GTA CTG AAG AAA TGG 1358 Pro Arg Ile His Leu Asp Glu Val Val Met Pro Val Leu Lys Lys Trp 255 260 265 270 TGT ATC TTC GAG CGC GAG GAC TTC ACC GGC GAG GGG GCT AAG CTG CGC 1406 Cys Ile Phe Glu Arg Glu Asp Phe Thr Gly Glu Gly Ala Lys Leu Arg 275 280 285 GAC GAG CTG GCC CTG GTG ATC AAG GAC CTC GAG CTG GCC TGC GAC AAG 1454 Asp Glu Leu Ala Leu Val Ile Lys Asp Leu Glu Leu Ala Cys Asp Lys 290 295 300 TTC GAG GTG TCC AAG CAA CGC CAA CTC GAC CGG GAA GCC CGT ACG GGC 1502 Phe Glu Val Ser Lys Gln Arg Gln Leu Asp Arg Glu Ala Arg Thr Gly 305 310 315 AAG AAG GTC AGC GCA CAC GAG CTG CAT AAA ACC GCT GGC AAA CTG GCG 1550 Lys Lys Val Ser Ala His Glu Leu His Lys Thr Ala Gly Lys Leu Ala 320 325 330 ATG AGC CGT CGT TAGCCCGGCG ACGATGCAGA GCGCGCAGCG CGATGAGCAG 1602 Met Ser Arg Arg 335 GAGGCGGGCA ATCCAACCCA GCCCGGCGAC GATGCAGAGC GCGCAGCGCG ATGAGCAGGA 1662 GGTGGGCAAT CCAACCCAGC CCGGCGTTG 1691 338 amino acids amino acid linear protein 4 Met Ser Ala Lys Leu Thr Asp Leu Gln Leu Leu His Glu Leu Glu Pro 1 5 10 15 Val Val Glu Lys Tyr Leu Asn Arg His Leu Ser Met His Lys Pro Trp 20 25 30 Asn Pro His Asp Tyr Ile Pro Trp Ser Asp Gly Lys Asn Tyr Tyr Ala 35 40 45 Leu Gly Gly Gln Asp Trp Asp Pro Asp Gln Ser Lys Leu Ser Asp Val 50 55 60 Ala Gln Val Ala Met Val Gln Asn Leu Val Thr Glu Asp Asn Leu Pro 65 70 75 80 Ser Tyr His Arg Glu Ile Ala Met Asn Met Gly Met Asp Gly Ala Trp 85 90 95 Gly Gln Trp Val Asn Arg Trp Thr Ala Glu Glu Asn Arg His Gly Ile 100 105 110 Ala Leu Arg Asp Tyr Leu Val Val Thr Arg Ser Val Asp Pro Val Glu 115 120 125 Leu Glu Lys Leu Arg Leu Glu Val Val Asn Arg Gly Phe Ser Pro Gly 130 135 140 Gln Asn His Gln Gly His Tyr Phe Ala Glu Ser Leu Thr Asp Ser Val 145 150 155 160 Leu Tyr Val Ser Phe Gln Glu Leu Ala Thr Arg Ile Ser His Arg Asn 165 170 175 Thr Gly Lys Ala Cys Asn Asp Pro Val Ala Asp Gln Leu Met Ala Lys 180 185 190 Ile Ser Ala Asp Glu Asn Leu His Met Ile Phe Tyr Arg Asp Val Ser 195 200 205 Glu Ala Ala Phe Asp Leu Val Pro Asn Gln Ala Met Lys Ser Leu His 210 215 220 Leu Ile Leu Ser His Phe Gln Met Pro Gly Phe Gln Val Pro Glu Phe 225 230 235 240 Arg Arg Lys Ala Val Val Ile Ala Val Gly Gly Val Tyr Asp Pro Arg 245 250 255 Ile His Leu Asp Glu Val Val Met Pro Val Leu Lys Lys Trp Cys Ile 260 265 270 Phe Glu Arg Glu Asp Phe Thr Gly Glu Gly Ala Lys Leu Arg Asp Glu 275 280 285 Leu Ala Leu Val Ile Lys Asp Leu Glu Leu Ala Cys Asp Lys Phe Glu 290 295 300 Val Ser Lys Gln Arg Gln Leu Asp Arg Glu Ala Arg Thr Gly Lys Lys 305 310 315 320 Val Ser Ala His Glu Leu His Lys Thr Ala Gly Lys Leu Ala Met Ser 325 330 335 Arg Arg 548 base pairs nucleic acid single Not Relevant other nucleic acid /desc = “NUCLEIC ACID” 5 GATCATCATC GGCCGGCTGC CGCGCAGGGC GCCGACACCG GCGAGTGCGG GCGCGAGGAT 60 CGGCCCCCAC CAGTTCGGCA GCTGCGTGTC GATGCGCTCC ACAATCCCGG GAAACAGCTC 120 GACCATTACC TCCTCAATAT GAGCCTCGAA AAACTTGCCG CTGTGCGCGG CGTCGTGGTG 180 AGCGCACACA ACAACTGTTA GCTGACCAGC AGGATCGGCG CTCTTACCGG TCTGTTCACC 240 GCATATCTGA ACGGACGGCT GGGAGCCACC CGCAAGCAAT TCATCGACTA CTGCGTCAAC 300 ATGTTGCTCA GCACCGCCGC CACCTACGCA CCGCACCGCG AGCGGGGAGA ATCCGAACAC 360 TCCATCCCAG CCGGGCCGCA CAACTGAGGA CGACTGGGGT TCACCCCACG CGGCCACCGG 420 GGCCCGCCGA TGCCAGCATC CTGCCCGCTG CTGGCAGCTC AACATGCCGC GCGAAGCCCA 480 AACTTGATGC TACCGAGAGA CACAGATATA TTGACTGCAA CCATTAGACA CAGATAACTG 540 GAGGCGCC 548 52 amino acids amino acid Not Relevant Not Relevant peptide 6 Glu Phe Tyr Lys Phe Leu Phe Thr Phe Leu Ala Met Ala Glu Lys Leu 1 5 10 15 Val Asn Phe Asn Ile Asp Glu Leu Val Thr Ser Phe Glu Ser His Asp 20 25 30 Ile Asp His Tyr Tyr Thr Glu Gln Lys Ala Met Glu Asn Val His Gly 35 40 45 Glu Thr Tyr Ala 50 52 amino acids amino acid Not Relevant Not Relevant peptide 7 Ile Phe Ile Ser Asn Leu Lys Tyr Gln Thr Leu Leu Asp Ser Ile Gln 1 5 10 15 Gly Arg Ser Pro Asn Val Ala Leu Leu Pro Leu Ile Ser Ile Pro Glu 20 25 30 Leu Glu Thr Trp Val Glu Thr Trp Ala Phe Ser Glu Thr Ile His Ser 35 40 45 Arg Ser Tyr Thr 50 52 amino acids amino acid Not Relevant Not Relevant peptide 8 Glu Thr Met Lys Val Val Ser Asn Phe Leu Glu Val Gly Glu Tyr Asn 1 5 10 15 Ala Ile Ala Ala Thr Gly Met Leu Trp Asp Ser Ala Gln Ala Ala Glu 20 25 30 Gln Lys Asn Gly Tyr Leu Ala Gln Val Leu Asp Glu Ile Arg His Thr 35 40 45 His Gln Cys Ala 50 52 amino acids amino acid single linear peptide 9 Glu Thr Met Lys Val Ile Ser Asn Phe Leu Glu Val Gly Glu Tyr Asn 1 5 10 15 Ala Ile Ala Ala Ser Ala Met Leu Trp Asp Ser Ala Thr Ala Ala Glu 20 25 30 Gln Lys Asn Gly Tyr Leu Ala Gln Val Leu Asp Glu Ile Arg His Thr 35 40 45 His Gln Cys Ala 50 52 amino acids amino acid single linear peptide 10 Asn Ala Leu Lys Leu Phe Leu Thr Ala Val Ser Pro Leu Glu Tyr Gln 1 5 10 15 Ala Phe Gln Gly Phe Ser Arg Val Gly Arg Gln Phe Ser Gly Ala Gly 20 25 30 Ala Arg Val Ala Cys Gln Met Gln Ala Ile Asp Glu Leu Arg His Val 35 40 45 Gln Thr Gln Val 50 52 amino acids amino acid single linear peptide 11 Ser Thr Leu Lys Ser His Tyr Gly Ala Ile Ala Val Gly Glu Tyr Ala 1 5 10 15 Ala Val Thr Gly Glu Gly Arg Met Ala Arg Phe Ser Lys Ala Pro Gly 20 25 30 Asn Arg Asn Met Ala Thr Phe Gly Met Met Asp Glu Leu Arg His Gly 35 40 45 Gln Leu Gln Leu 50 54 amino acids amino acid single linear peptide 12 Leu Val Gly Asp Met Ile Thr Glu Glu Ala Leu Pro Thr Tyr Gln Thr 1 5 10 15 Met Leu Asn Thr Leu Asp Gly Val Arg Asp Glu Thr Gly Ala Ser Pro 20 25 30 Thr Ser Trp Ala Ile Trp Thr Arg Ala Trp Thr Ala Glu Glu Asn Arg 35 40 45 His Gly Asp Leu Leu Asn 50 54 amino acids amino acid single linear peptide 13 Leu Val Gly Asp Met Ile Thr Glu Glu Ala Leu Pro Thr Tyr Gln Thr 1 5 10 15 Met Leu Asn Thr Leu Asp Gly Val Arg Asp Glu Thr Gly Ala Ser Pro 20 25 30 Thr Pro Trp Ala Ile Trp Thr Arg Ala Trp Thr Ala Glu Glu Asn Arg 35 40 45 His Gly Asp Leu Leu Asn 50 54 amino acids amino acid single linear peptide 14 Leu Val Gly Asp Met Ile Thr Glu Glu Ala Leu Pro Thr Tyr Gln Thr 1 5 10 15 Met Leu Asn Thr Leu Asp Gly Val Arg Asp Glu Thr Gly Ala Ser Leu 20 25 30 Thr Pro Trp Ala Val Trp Thr Arg Ala Trp Thr Ala Glu Glu Asn Arg 35 40 45 His Gly Asp Leu Leu His 50 54 amino acids amino acid single linear peptide 15 Leu Val Gly Asp Met Ile Thr Glu Glu Ala Leu Pro Thr Tyr Gln Thr 1 5 10 15 Met Leu Asn Thr Leu Asp Gly Ala Lys Asp Glu Thr Gly Ala Ser Pro 20 25 30 Thr Ser Trp Ala Val Trp Thr Arg Ala Trp Thr Ala Glu Glu Asn Arg 35 40 45 His Gly Asp Leu Leu Asn 50 54 amino acids amino acid single linear peptide 16 Leu Val Gly Asp Met Ile Thr Glu Glu Ala Leu Pro Thr Tyr Gln Thr 1 5 10 15 Met Leu Asn Thr Leu Asp Gly Val Arg Asp Glu Thr Gly Ala Ser Pro 20 25 30 Thr Ser Trp Ala Ile Trp Thr Arg Ala Trp Thr Ala Glu Glu Asn Arg 35 40 45 His Gly Asp Leu Leu Asn 50 54 amino acids amino acid single linear peptide 17 Leu Ile Gly Asp Met Ile Thr Glu Glu Ala Leu Pro Thr Tyr Gln Thr 1 5 10 15 Met Ile Asn Thr Leu Asp Gly Val Arg Asp Glu Thr Gly Ala Thr Val 20 25 30 Thr Pro Trp Ala Ile Trp Thr Arg Ala Trp Thr Ala Glu Glu Asn Arg 35 40 45 His Gly Asp Leu Leu Asn 50 54 amino acids amino acid single linear peptide 18 Leu Val Gly Asp Met Ile Thr Glu Glu Ala Leu Pro Thr Tyr Gln Thr 1 5 10 15 Met Leu Asn Thr Leu Asp Gly Val Arg Asp Glu Thr Gly Ala Ser Leu 20 25 30 Thr Pro Trp Ala Ile Trp Thr Arg Ala Trp Thr Ala Glu Glu Asn Arg 35 40 45 His Gly Asp Leu Leu Asn 50 54 amino acids amino acid single linear peptide 19 Leu Val Gly Asp Met Ile Thr Glu Glu Ala Leu Pro Thr Tyr Met Ser 1 5 10 15 Met Leu Asn Arg Cys Asp Gly Ile Lys Asp Asp Thr Gly Ala Gln Pro 20 25 30 Thr Ser Trp Ala Thr Trp Thr Arg Ala Trp Thr Ala Glu Glu Asn Arg 35 40 45 His Gly Asp Leu Leu Asn 50 54 amino acids amino acid single linear peptide 20 Ser Asp Val Ala Gln Val Ala Met Val Gln Asn Leu Val Thr Glu Asp 1 5 10 15 Asn Leu Pro Ser Tyr His Arg Glu Ile Ala Met Asn Met Gly Met Asp 20 25 30 Gly Ala Trp Gly Gln Trp Val Asn Arg Trp Thr Ala Glu Glu Asn Arg 35 40 45 His Gly Ile Ala Leu Arg 50 52 amino acids amino acid single linear peptide 21 Glu Lys Ile Leu Val Phe Leu Leu Ile Glu Gly Ile Phe Phe Ile Ser 1 5 10 15 Ser Phe Tyr Ser Ile Ala Leu Leu Arg Val Arg Gly Leu Met Pro Gly 20 25 30 Ile Cys Leu Ala Asn Asn Tyr Ile Ser Arg Asp Glu Leu Leu His Thr 35 40 45 Arg Ala Ala Ser 50 52 amino acids amino acid single linear peptide 22 Leu Cys Leu Met Ser Val Asn Ala Leu Glu Ala Ile Arg Phe Tyr Val 1 5 10 15 Ser Phe Ala Cys Ser Phe Ala Phe Ala Glu Arg Glu Leu Met Glu Gly 20 25 30 Asn Ala Lys Ile Ile Arg Leu Ile Ala Arg Asp Glu Ala Leu His Leu 35 40 45 Thr Gly Thr Gln 50 52 amino acids amino acid single linear peptide 23 Cys Ser Leu Asn Leu Gln Leu Val Gly Glu Ala Cys Phe Thr Asn Pro 1 5 10 15 Leu Ile Val Ala Val Thr Glu Trp Ala Ala Ala Asn Gly Asp Glu Ile 20 25 30 Thr Pro Thr Val Phe Leu Ser Ile Glu Thr Asp Glu Leu Arg His Met 35 40 45 Ala Asn Gly Tyr 50 52 amino acids amino acid single linear peptide 24 Cys Ser Val Asn Leu Gln Leu Val Gly Asp Thr Cys Phe Thr Asn Pro 1 5 10 15 Leu Ile Val Ala Val Thr Glu Trp Ala Ile Gly Asn Gly Asp Glu Ile 20 25 30 Thr Pro Thr Val Phe Leu Ser Val Glu Thr Asp Glu Leu Arg His Met 35 40 45 Ala Asn Gly Tyr 50 52 amino acids amino acid single linear peptide 25 Phe Leu Thr Ala Val Ser Phe Ser Phe Glu Tyr Val Leu Thr Asn Leu 1 5 10 15 Leu Phe Val Pro Phe Met Ser Gly Ala Ala Tyr Asn Gly Asp Met Ala 20 25 30 Thr Val Thr Phe Gly Phe Ser Ala Gln Ser Asp Glu Ala Arg His Met 35 40 45 Thr Leu Gly Leu 50 52 amino acids amino acid single linear peptide 26 Val Ala Ile Met Leu Thr Phe Ser Phe Glu Thr Gly Phe Thr Asn Met 1 5 10 15 Gln Phe Leu Gly Leu Ala Ala Asp Ala Ala Glu Ala Gly Asp Tyr Thr 20 25 30 Phe Ala Asn Leu Ile Ser Ser Ile Gln Thr Asp Glu Ser Arg His Ala 35 40 45 Gln Gln Gly Gly 50 52 amino acids amino acid single linear peptide 27 Tyr Leu Gly Phe Ile Tyr Thr Ser Phe Gln Glu Arg Ala Thr Phe Ile 1 5 10 15 Ser His Gly Asn Thr Ala Arg Gln Ala Lys Glu His Gly Asp Ile Lys 20 25 30 Leu Ala Gln Ile Cys Gly Thr Ile Ala Ala Asp Glu Lys Arg His Glu 35 40 45 Thr Ala Tyr Thr 50 52 amino acids amino acid single linear peptide 28 Tyr Leu Gly Phe Ile Tyr Thr Ser Phe Gln Glu Arg Ala Thr Phe Ile 1 5 10 15 Ser His Gly Asn Thr Ala Arg Leu Ala Lys Glu His Gly Asp Ile Lys 20 25 30 Leu Ala Gln Ile Cys Gly Thr Ile Thr Ala Asp Glu Lys Arg His Glu 35 40 45 Thr Ala Tyr Thr 50 52 amino acids amino acid single linear peptide 29 Tyr Leu Gly Phe Ile Tyr Thr Ser Phe Gln Glu Arg Ala Thr Phe Val 1 5 10 15 Ser His Gly Asn Thr Ala Arg His Ala Lys Asp His Gly Asp Val Lys 20 25 30 Leu Ala Gln Ile Cys Gly Thr Ile Ala Ser Asp Glu Lys Arg His Glu 35 40 45 Thr Ala Tyr Thr 50 52 amino acids amino acid single linear peptide 30 Tyr Leu Gly Phe Val Tyr Thr Ser Phe Gln Glu Arg Ala Thr Phe Val 1 5 10 15 Ser His Gly Asn Ser Ala Arg Leu Ala Lys Glu His Gly Asp Leu Lys 20 25 30 Met Ala Gln Ile Cys Gly Ile Ile Ala Ser Asp Glu Lys Arg His Glu 35 40 45 Thr Ala Tyr Thr 50 52 amino acids amino acid single linear peptide 31 Tyr Leu Gly Phe Ile Tyr Thr Ser Phe Gln Glu Arg Ala Thr Phe Ile 1 5 10 15 Ser His Gly Asn Thr Ala Arg Gln Ala Lys Glu His Gly Asp Leu Lys 20 25 30 Leu Ala Gln Ile Cys Gly Thr Ile Ala Ala Asp Glu Lys Arg His Glu 35 40 45 Thr Ala Tyr Thr 50 52 amino acids amino acid single linear peptide 32 Tyr Leu Gly Phe Val Tyr Thr Ser Leu Arg Lys Gly Val Thr Phe Val 1 5 10 15 Ser His Gly Asn Thr Ala Arg Leu Ala Lys Glu His Gly Asp Met Lys 20 25 30 Leu Ala Gln Ile Cys Gly Ser Ile Ala Ala Asp Glu Lys Arg His Glu 35 40 45 Thr Ala Tyr Thr 50 52 amino acids amino acid single linear peptide 33 Tyr Leu Gly Phe Ile Tyr Thr Ser Phe Gln Glu Arg Ala Thr Phe Ile 1 5 10 15 Ser His Gly Asn Thr Ala Arg Leu Ala Lys Asp His Gly Asp Met Lys 20 25 30 Leu Ala Gln Ile Cys Gly Ile Ile Ala Ala Asp Glu Lys Arg His Glu 35 40 45 Thr Ala Tyr Thr 50 52 amino acids amino acid single linear peptide 34 Tyr Met Gly Phe Ile Tyr Thr Ser Phe Gln Glu Arg Ala Thr Phe Ile 1 5 10 15 Ser His Ala Asn Thr Ala Lys Leu Ala Gln His Tyr Gly Asp Lys Asn 20 25 30 Leu Ala Gln Val Cys Gly Asn Ile Ala Ser Asp Glu Lys Arg His Ala 35 40 45 Thr Ala Tyr Thr 50 49 amino acids amino acid single linear peptide 35 Thr Asp Ser Val Leu Tyr Val Ser Phe Gln Glu Leu Ala Thr Arg Ile 1 5 10 15 Ser His Arg Asn Thr Gly Lys Ala Cys Asn Asp Pro Val Ala Asp Gln 20 25 30 Leu Met Ala Lys Ile Ser Ala Asp Glu Asn Leu His Met Ile Phe Tyr 35 40 45 Arg 1600 base pairs nucleic acid Not Relevant Not Relevant other nucleic acid /desc = “NUCLEIC ACID” CDS 549..1562 36 GATCATCATC GGCCGGCTGC CGCGCAGGGC GCCGACACCG GCGAGTGCGG GCGCGAGGAT 60 CGGCCCCCAC CAGTTCGGCA GCTGCGTGTC GATGCGCTCC ACAATCCCGG GAAACAGCTC 120 GACCATTACC TCCTCAATAT GAGCCTCGAA AAACTTGCCG CTGTGCGCGG CGTCGTGGTG 180 AGCGCACACA ACAACTGTTA GCTGACCAGC AGGATCGGCG CTCTTACCGG TCTGTTCACC 240 GCATATCTGA ACGGACGGCT GGGAGCCACC CGCAAGCAAT TCATCGACTA CTGCGTCAAC 300 ATGTTGCTCA GCACCGCCGC CACCTACGCA CCGCACCGCG AGCGGGGAGA ATCCGAACAC 360 TCCATCCCAG CCGGGCCGCA CAACTGAGGA CGACTGGGGT TCACCCCACG CGGCCACCGG 420 CGCCCGCCGA TGCCAGCATC CTGCCCGCTG CTGGCAGCTC AACATGCCGC GCGAAGCCCA 480 AACTTGATGC TACCGAGAGA CACAGATATA TTGACTGCAA CCATTAGACA CAGATAACTG 540 GAGGCGCC ATG TCA GCC AAG CTG ACC GAC CTG CAG CTG CTG CAC GAA CTT 590 Met Ser Ala Lys Leu Thr Asp Leu Gln Leu Leu His Glu Leu 340 345 350 GAA CCG GTC GTC GAG AAG TAC CTG AAC CGG CAC CTG AGC ATG CAC AAG 638 Glu Pro Val Val Glu Lys Tyr Leu Asn Arg His Leu Ser Met His Lys 355 360 365 CCC TGG AAC CCG CAC GAC TAC ATC CCG TGG TCG GAC GGG AAG AAC TAC 686 Pro Trp Asn Pro His Asp Tyr Ile Pro Trp Ser Asp Gly Lys Asn Tyr 370 375 380 TAC GCG CTC GGC GGG CAG GAT TGG GAC CCC GAC CAG AGC AAG CTT TCT 734 Tyr Ala Leu Gly Gly Gln Asp Trp Asp Pro Asp Gln Ser Lys Leu Ser 385 390 395 400 GAT GTC GCC CAG GTG GCG ATG GTG CAG AAC CTG GTC ACC GAG GAC AAC 782 Asp Val Ala Gln Val Ala Met Val Gln Asn Leu Val Thr Glu Asp Asn 405 410 415 CTG CCG TCG TAT CAC CGC GAG ATC GCG ATG AAC ATG GGC ATG GAC GGC 830 Leu Pro Ser Tyr His Arg Glu Ile Ala Met Asn Met Gly Met Asp Gly 420 425 430 GCG TGG GGG CAG TGG GTC AAC CGT TGG ACC GCC GAG GAG AAT CGG CAC 878 Ala Trp Gly Gln Trp Val Asn Arg Trp Thr Ala Glu Glu Asn Arg His 435 440 445 GGC ATC GCG CTG CGC GAC TAC CTG GTG GTG ACC CGA TCG GTC GAC CCT 926 Gly Ile Ala Leu Arg Asp Tyr Leu Val Val Thr Arg Ser Val Asp Pro 450 455 460 GTC GAG TTG GAG AAA CTT CGC CTC GAG GTA GTC AAC CGG GGC TTC AGC 974 Val Glu Leu Glu Lys Leu Arg Leu Glu Val Val Asn Arg Gly Phe Ser 465 470 475 480 CCA GGC CAA AAC CAC CAG GGC CAC TAT TTC GCG GAG AGC CTC ACC GAC 1022 Pro Gly Gln Asn His Gln Gly His Tyr Phe Ala Glu Ser Leu Thr Asp 485 490 495 TCC GTC CTC TAT GTC AGT TTC CAG GAA CTG GCA ACC CGG ATT TCG CAC 1070 Ser Val Leu Tyr Val Ser Phe Gln Glu Leu Ala Thr Arg Ile Ser His 500 505 510 CGC AAT ACC GGC AAG GCA TGT AAC GAC CCC GTC GCC GAC CAG CTC ATG 1118 Arg Asn Thr Gly Lys Ala Cys Asn Asp Pro Val Ala Asp Gln Leu Met 515 520 525 GCC AAG ATC TCG GCA GAC GAG AAT CTG CAC ATG ATC TTC TAC CGC GAC 1166 Ala Lys Ile Ser Ala Asp Glu Asn Leu His Met Ile Phe Tyr Arg Asp 530 535 540 GTC AGC GAG GCC GCG TTC GAC CTC GTG CCC AAC CAG GCC ATG AAG TCG 1214 Val Ser Glu Ala Ala Phe Asp Leu Val Pro Asn Gln Ala Met Lys Ser 545 550 555 560 CTG CAC CTG ATT TTG AGC CAC TTC CAG ATG CCC GGC TTC CAA GTA CCC 1262 Leu His Leu Ile Leu Ser His Phe Gln Met Pro Gly Phe Gln Val Pro 565 570 575 GAG TTC CGG CGC AAA GCC GTG GTC ATC GCC GTC GGG GGT GTC TAC GAC 1310 Glu Phe Arg Arg Lys Ala Val Val Ile Ala Val Gly Gly Val Tyr Asp 580 585 590 CCG CGC ATC CAC CTC GAC GAA GTC GTC ATG CCG GTA CTG AAG AAA TGG 1358 Pro Arg Ile His Leu Asp Glu Val Val Met Pro Val Leu Lys Lys Trp 595 600 605 TGT ATC TTC GAG CGC GAG GAC TTC ACC GGC GAG GGG GCT AAG CTG CGC 1406 Cys Ile Phe Glu Arg Glu Asp Phe Thr Gly Glu Gly Ala Lys Leu Arg 610 615 620 GAC GAG CTG GCC CTG GTG ATC AAG GAC CTC GAG CTG GCC TGC GAC AAG 1454 Asp Glu Leu Ala Leu Val Ile Lys Asp Leu Glu Leu Ala Cys Asp Lys 625 630 635 640 TTC GAG GTG TCC AAG CAA CGC CAA CTC GAC CGG GAA GCC CGT ACG GGC 1502 Phe Glu Val Ser Lys Gln Arg Gln Leu Asp Arg Glu Ala Arg Thr Gly 645 650 655 AAG AAG GTC AGC GCA CAC GAG CTG CAT AAA ACC GCT GGC AAA CTG GCG 1550 Lys Lys Val Ser Ala His Glu Leu His Lys Thr Ala Gly Lys Leu Ala 660 665 670 ATG AGC CGT CGT TAGCCCGGCG ACGATGCAGA GCGCGCAGCG CGATGAGC 1600 Met Ser Arg Arg 675 338 amino acids amino acid linear protein 37 Met Ser Ala Lys Leu Thr Asp Leu Gln Leu Leu His Glu Leu Glu Pro 1 5 10 15 Val Val Glu Lys Tyr Leu Asn Arg His Leu Ser Met His Lys Pro Trp 20 25 30 Asn Pro His Asp Tyr Ile Pro Trp Ser Asp Gly Lys Asn Tyr Tyr Ala 35 40 45 Leu Gly Gly Gln Asp Trp Asp Pro Asp Gln Ser Lys Leu Ser Asp Val 50 55 60 Ala Gln Val Ala Met Val Gln Asn Leu Val Thr Glu Asp Asn Leu Pro 65 70 75 80 Ser Tyr His Arg Glu Ile Ala Met Asn Met Gly Met Asp Gly Ala Trp 85 90 95 Gly Gln Trp Val Asn Arg Trp Thr Ala Glu Glu Asn Arg His Gly Ile 100 105 110 Ala Leu Arg Asp Tyr Leu Val Val Thr Arg Ser Val Asp Pro Val Glu 115 120 125 Leu Glu Lys Leu Arg Leu Glu Val Val Asn Arg Gly Phe Ser Pro Gly 130 135 140 Gln Asn His Gln Gly His Tyr Phe Ala Glu Ser Leu Thr Asp Ser Val 145 150 155 160 Leu Tyr Val Ser Phe Gln Glu Leu Ala Thr Arg Ile Ser His Arg Asn 165 170 175 Thr Gly Lys Ala Cys Asn Asp Pro Val Ala Asp Gln Leu Met Ala Lys 180 185 190 Ile Ser Ala Asp Glu Asn Leu His Met Ile Phe Tyr Arg Asp Val Ser 195 200 205 Glu Ala Ala Phe Asp Leu Val Pro Asn Gln Ala Met Lys Ser Leu His 210 215 220 Leu Ile Leu Ser His Phe Gln Met Pro Gly Phe Gln Val Pro Glu Phe 225 230 235 240 Arg Arg Lys Ala Val Val Ile Ala Val Gly Gly Val Tyr Asp Pro Arg 245 250 255 Ile His Leu Asp Glu Val Val Met Pro Val Leu Lys Lys Trp Cys Ile 260 265 270 Phe Glu Arg Glu Asp Phe Thr Gly Glu Gly Ala Lys Leu Arg Asp Glu 275 280 285 Leu Ala Leu Val Ile Lys Asp Leu Glu Leu Ala Cys Asp Lys Phe Glu 290 295 300 Val Ser Lys Gln Arg Gln Leu Asp Arg Glu Ala Arg Thr Gly Lys Lys 305 310 315 320 Val Ser Ala His Glu Leu His Lys Thr Ala Gly Lys Leu Ala Met Ser 325 330 335 Arg Arg 

What is claimed is:
 1. A purified polypeptide comprising the amino acid sequence of SEQ ID NO:4, wherein the polypeptide is recognized by antibodies present in the sera of patients infected by bacteria of the Mycobacterium species.
 2. The polypeptide according to claim 1, wherein the Mycobacterium species is Mycobacterium tuberculosis.
 3. The polypeptide according to claim 1, wherein the Mycobacterium species is Mycobacterium bovis. 